WO1998001466A1 - Imprint polypeptides covalently cross-linked and having a fixed and stabilised arrangement, process for the preparation and use thereof - Google Patents
Imprint polypeptides covalently cross-linked and having a fixed and stabilised arrangement, process for the preparation and use thereof Download PDFInfo
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- WO1998001466A1 WO1998001466A1 PCT/DE1997/001448 DE9701448W WO9801466A1 WO 1998001466 A1 WO1998001466 A1 WO 1998001466A1 DE 9701448 W DE9701448 W DE 9701448W WO 9801466 A1 WO9801466 A1 WO 9801466A1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/282—Porous sorbents
- B01J20/285—Porous sorbents based on polymers
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3852—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36 using imprinted phases or molecular recognition; using imprinted phases
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2650/00—Assays involving polymers whose constituent monomers bore biological functional groups before polymerization, i.e. vinyl, acryl derivatives of amino acids, sugars
Definitions
- the invention relates in its conformation to fixed and stabilized, covalently cross-linked imprint polypeptides (embossed polypeptides), e.g. Proteins that can be used in the catalytic synthesis, chromatography and analysis of chiral compounds as well as in biosensor technology for the specific recognition of molecules, processes for their production and their use.
- imprint polypeptides e.g. Proteins that can be used in the catalytic synthesis, chromatography and analysis of chiral compounds as well as in biosensor technology for the specific recognition of molecules, processes for their production and their use.
- Polymers with the ability to selectively recognize molecules are of the greatest industrial importance in biotechnology and chemistry. These include the enzymes, antibodies and receptors that belong to the class of proteins. Because of their three-dimensional molecular structure (conformation), these polymers have complementary regions for certain molecules (substrates, antigens, hormones, etc.), which are referred to as binding sites. Since native polymers often bind commercially interesting molecules inadequately or not at all or the conformation necessary for binding is not sufficiently stable under the conditions of use, methods have been developed to produce tailor-made polymers which have the desired complementarity (K. Mosbach and O. Ramström, Biotechnology, Vol. 14 (1996), 163-170).
- Goldstein, Methods Enzymol. 19 (1970), 935-962, and Fritz et al., Angew. Chem. 78, 1966, 775 describe a method for immobilizing proteins using cyclic anhydrides for the covalent coupling.
- the process consists of first copolymerizing an anhydride into a carrier matrix with other monomers and then coupling the protein to the finished carrier.
- Both variants are based on the principle that a specially selected molecule, hereinafter referred to as the print molecule (embossing molecule), acts as a shaping agent (embosser) and for the desired complementarity in the polymer and thus new property is responsible.
- Figures 1 and 2 schematically explain these two variants.
- Variant 1 for the production of customized (embossed) polymers is carried out in such a way that a copolymerization of selected monomers is carried out in an organic solvent in the presence of the print molecule (see FIG. 1).
- the print molecule a) is dissolved directly in an organic solvent system together with a monomer (non-covalent method) or b) after derivatization with a monomer (covalent method), then a crosslinker and a radical initiator are added and the polymerization is started.
- the print molecule must then be extracted from the resulting polymer under suitable conditions so that the new binding sites generated in the polymer are released.
- the polymer produced in this way has selective recognition sites for the print molecule or for structurally similar molecules (see FIG. 1).
- the numbers mean functional groups.
- the polymer is first dissolved together with the print molecule in an aqueous solvent system and then precipitated by adding additives, for example organic solvents.
- additives for example organic solvents.
- the print molecule and the polymer interact with one another via non-covalent interactions in such a way that the conformation of the polymer is changed and the polymer thereby has a new property which is inherently predetermined by the print molecule.
- the process of variant 2 described up to this point is hereinafter referred to as I printing (embossing), the resulting polymer as an imprint polymer (embossed polymer); eg imprint polypeptide.
- the new property of the imprint polymer is reversible. It only remains as long as it is used under conditions which prevent the reformation to the originally thermodynamically more favorable conformation. Therefore, the polymers obtained by variant 2, which by Mosbach et al. loc. cit., Stähl et al., Biotechnol. Lett. 12: 161-166 (1990); Stähl et al., J. Am. Chem. Soc. 113 (1991), 9366-9368 and Klibanov et al., J. Biol. Chem. 263 (1988), 11624-11626 and Dabulis and Klibanow, Biotechnol. Bioeng. 39 (1992), 176-185, can only be used in organic solvents.
- Fig. 2 There are two options for imprinting (see Fig. 2).
- the first possibility resulted in the use of bovine serum albumin as a polypeptide and L-malic acid as a print molecule, an imprint polypeptide, which was selective in chromatographic studies for the print molecule L-malic acid (Dabulis and Klibanow, loc. Cit).
- subtilisin Rairel and Klibanow, loc. cit.
- ⁇ -chymotrypsin Stähl et al., loc. cit.
- the polypeptide ⁇ -chymotrypsin for example, has the property of hydrolyzing the (Z -) enantiomer of the N-acetyl-tryptophanethyl ester (N-Ac-TrpEE) in an aqueous solvent system or in an organic solvent system composed of N-acetyl-L-tryptophan (N-Ac-Trp) and ethanol.
- the () enantiomer of N-Ac-TrpEE cannot be hydrolyzed or synthesized.
- this polypeptide ⁇ -chymotrypsin is imprinted with the print molecule N-Ac- (£>) - Trp, it can then be synthesized in organic solvent from N-Ac- (Z)) - TrpEE from N-Ac- ( -D) -Trp and ethanol catalyze, since the conformation of the polypeptide was specifically changed by the print molecule (Stähl et al., Loc. Cit.). However, the change in conformation is reversible. Therefore, the imprint polypeptide cannot be used in the aqueous solvent system for the hydrolysis of the N-Ac - (- D) -TrpEE. The new property is lost when the imprint polypeptide comes into contact with aqueous solvent systems.
- a limitation of the imprinting method carried out so far is that the imprint polypeptides are labile and have or retain their imprinted (embossed) new properties exclusively in the organic solvent system.
- the conformation of the imprint polypeptides is based on sensitive, non-covalent bonds. As soon as they come into contact with aqueous solvent systems, the properties induced by imprinting are no longer detectable and irreversibly lost, since the induced change in conformation in aqueous systems is energetically unfavorable. If covalent fixation and stabilization of the conformation of the imprint polypeptides were possible, they could then also be used in an aqueous environment. The imprinted conformation would be fixed covalently and would not be lost without breaking the bonded bonds.
- the invention is therefore based on the object of making available, in its conformation, fixed and stabilized, covalently cross-linked imprint polypeptides which can also be used in aqueous media.
- the new or expanded catalysis and / or binding properties of the imprint polypeptides should be fixed and stabilized. This is the loss, i.e. the reversibility, the imprinted properties) of the polypeptides no longer exist in aqueous media.
- the imprint polypeptides can then be used for the first time in catalytic, chromatographic and / or analytical processes that are carried out in aqueous media.
- step (B) imprinting the polypeptide obtained in step (A) with a print molecule in an aqueous medium
- step (C) Precipitation of the polypeptide / print molecule mixture obtained in step (B) by adding an additive suitable for precipitation and / or
- step (D) copolymerizing the imprint polypeptide obtained in step (C) in an organic solvent with a crosslinker which is copolymerizable with the polymerizable groups containing unsaturated bonds.
- the covalently cross-linked imprint polypeptides which are fixed and stabilized in their conformation can be obtained by the following steps: (A) covalently introducing polymerizable groups containing unsaturated bonds into a polypeptide in an aqueous medium;
- step (B) imprinting the polypeptide obtained in step (A) with a print molecule in an aqueous medium
- step (C) copolymerization of the polypeptide obtained in step (B) in an aqueous medium with a crosslinker which is copolymerizable with the polymerizable groups containing unsaturated bonds.
- step (B) before step (A) or steps (A) and (B) simultaneously.
- Figure 3 is a schematic representation of the method for fixing and stabilizing polypeptides.
- Figure 4 is a schematic representation of the method for fixing
- FIG. 5 shows a review of the “tailor-made”
- the method according to the invention comprises the selective introduction of polymerizable groups containing unsaturated bonds into an optionally imprinted (embossed) polypeptide, for example a protein.
- the process according to the invention comprises the further step (D) that selectively on the groups of the derivatized polypeptides containing unsaturated bonds a covalent crosslinking by copolymerization with a crosslinker, For example, a divinylated compound takes place, which fixes and stabilizes the desired polypeptide conformation covalently.
- the covalently cross-linked imprint polypeptide is then both in stable in aqueous as well as in organic solvent systems and the imprinted property, for example a changed catalysis and / or affinity property, can be used for commercial applications (see FIGS. 3 and 4).
- polypeptides with, for example, alkyl, aryl, OH, NH 2 , SH, and / or COOH groups are suitable, which, on the one hand, are hydrophobic with the print molecule via non-covalent bonds, for example ion bonds, hydrogen bonds Interactions, van der Waals forces, metal chelate complexes, interact and, secondly, before, during or after imprinting can be derivatized covalently with groups that contain polymerizable, unsaturated bonds.
- polypeptides with OH, NH 2 and / or SH groups are used as starting compounds.
- native proteins are used as polypeptides, preferably enzymes, in particular ⁇ -glucosidase or ⁇ -chymotrypsin.
- Preferred enzymes are selected from oxido reductases such as D- and L-amino acid oxidases, alcohol dehydrogenase, glucose oxidase and formate dehydrogenase. Further preferred enzymes are selected from hydrolases, such as proteases, peptidases (e.g. Rennin), amylases (e.g. ⁇ -amylase, ⁇ -amylase, glucoamylase, ⁇ -galactosidase), glycosidases, acyiases, lipases and esterases.
- oxido reductases such as D- and L-amino acid oxidases, alcohol dehydrogenase, glucose oxidase and formate dehydrogenase.
- hydrolases such as proteases, peptidases (e.g. Rennin), amylases (e.g. ⁇ -amylase, ⁇ -amylase, glucoamylase, ⁇ -galact
- preferred enzymes can be selected from isomerases such as glucose isomerase and amino acid racemases.
- Polymerases such as DNA polymerases, transferases such as phosphotransferases or ligases such as DNA ligases can be used as further preferred enzymes.
- the introduction of the polymerizable groups containing unsaturated bonds is carried out in an aqueous medium.
- the pH of the aqueous medium is preferably about 3 to 12, preferably about 5 to 8 and in particular about 6 to 8.
- the aqueous medium can contain a buffer system.
- the buffer system is preferably selected from a potassium phosphate buffer, a citric acid phosphate buffer, Tris buffer (tris (hydroxymethyl) aminomethane) or a sodium phosphate buffer.
- the process according to the invention is preferably characterized in that the introduction of the polymerizable groups containing unsaturated bonds, preferably vinyl groups, has to be carried out in a polypeptide in a selective manner.
- Some of the functional groups of the polypeptides (OH, NH 2 , SH, COOH groups) which are to interact with a print molecule in the aqueous solvent system during imprinting should not be derivatized. This can be controlled, for example, by the molar ratio of polypeptide / olefinic compound.
- the degree of derivatization is determined by the respective primary sequence of the polypeptide, the amino acid composition, the temperature and the pH.
- the optimal degree of derivatization for maintaining the imprinted property can easily be checked by comparing the underivatized imprint polypeptide with the differently derivatized imprint polypeptides with regard to the desired property (s) (FIG. 5).
- Preferred polypeptide / olefinic compound molar ratios range from about 1: 5 to 1: 300.
- olefinic compounds or mixtures thereof are selected from the group consisting of reactive analogs or derivatives of the general formula (I)
- the radicals R ,, R 2 , R 3 and R 4 independently of one another hydrogen atoms, carboxyl radicals, cyclic or linear, substituted or unsubstituted, saturated or unsaturated alkyl radicals, alkoxy radicals or carboxyalkyl radicals with preferably up to 10, more preferably up to 6, in particular 1 or 2 carbon atoms or substituted or unsubstituted aryl radicals or carboxyaryl radicals, with the proviso that at least one of the radicals R 1 , R 2 , R 3 and R 4 is independently a carboxyl, carboxyalkyl or carboxyaryl radical.
- radicals RR 2 , R 3 and R 4 are, independently of one another, a carboxyl, carboxyalkyl or carboxyaryl radical. especially carboxyl or Carboxyalkyl radicals with in particular 1 or 2 carbon atoms.
- the radicals RR 2 , R 3 or R which are not a carboxyl, carboxyalkyl or carboxyaryl radical are preferably, independently of one another, hydrogen atoms or alkyl radicals, more preferably ethyl or methyl radicals, in particular methyl radicals.
- R 1? R 2 , R 3 and R are substituted alkyl, carboxyalkyl, alkoxy, aryl or carboxyaryl radicals
- the substituents are preferably selected from the group consisting of halogen atoms, nitro, amido, carboxyl, ester and alkoxy radicals preferably up to 10, more preferably up to 5 and in particular 1 or 2 carbon atoms.
- the radicals RR 2 , R 3 and R 4 are unsaturated alkyl, carboxyalkyl or alkoxy radicals, they preferably contain up to 5, more preferably up to 3 and in particular an unsaturated bond.
- radicals R ,, R, R 3 and R are substituted or unsubstituted aryl radicals or carboxyaryl radicals
- the aryl radicals are preferably phenyl or naphthyl radicals, the substituents preferably being selected from those for substituted alkyl, carboxyalkyl, alkoxy, aryl - Or carboxyaryl groups called.
- compounds selected from the group consisting of itaconic, maleic, citracon, croton, methacrylic and acrylic acid are used.
- olefinic cyclic or non-cyclic anhydrides such as itacon, maleic, citraconic or crotonic, acrylic or methacrylic anhydride, or the acid halides, in particular the acid chlorides of the olefinic compounds mentioned, are used for the derivatization.
- ISA itaconic anhydride
- aqueous medium preferably in the pH range from about 3-12, which binds selectively and covalently to OH, NH 2 and / or SH groups (cf. Tab. 3).
- the number of ISA bonds is determined by the individual primary sequence of the polypeptide, the temperature and the pH.
- the optimal percentage of possible derivatization for maintaining the imprinted property is checked.
- the non-derivatized imprint polypeptide is compared with the imprint polypeptides derivatized to different degrees in terms of the desired property (s) (FIG. 5).
- polyols such as polyethylene glycols, sorbitol, sucrose, glucose and / or glycerol or salts
- step (A) polyols, such as polyethylene glycols, sorbitol, sucrose, glucose and / or glycerol or salts
- step (A) ammonium, alkali or alkaline earth metal sulfates, phosphates or halides, such as (NH 4 ) 2 SO 4 , Na 2 SO 4 , MgSO 4 , Na 3 PO 4 , CaHP0 4 , (NH 4 ) H 2 PO 4 , NH 4 C1, NaCI, KC1 and CaCl 2 .
- This process step thus has many degrees of freedom in order to obtain a functionally active and derivatized imprint polypeptide as a result.
- the process according to the invention is also characterized in that the polypeptide is incubated with a print molecule in an aqueous medium before, during or after the covalent introduction of polymerizable groups containing unsaturated bonds.
- the print molecule and the polypeptide form a polypeptide / print molecule mixture, the print molecule and the polypeptide interacting via non-covalent interactions. This changes the conformation of the polypeptide, which thus has new and / or changed properties.
- a compound which has a high structural and / or chiral similarity to the subsequent substrate of the finished, covalently cross-linked imprint polypeptide which is fixed and stabilized in its conformation or which is identical to the subsequent substrate can preferably be used as the print molecule.
- ⁇ -chymotrypsin is used as the polypeptide and N-Ac- (Z) -ryptophan, N-Ac- (D) -tyrosine or N-Ac- (D) -phenylalanine is used as the print molecule.
- bovine serum albumin is used as the polypeptide and L-malic acid is used as the print molecule, or D- or L-amino acid oxidase is used as the polypeptide and natural or non-natural (D) or (L) - amino acids are used as the print molecule.
- the inventive method is characterized in that the polypeptide / print molecule mixture dissolved in an aqueous medium is precipitated.
- Precipitation can be carried out by adding additives which are suitable for precipitating the polypeptide / print molecule mixture in an aqueous medium.
- water-miscible organic solvents are used as additives for the precipitation, preferably acetone, methyl ethyl ketone, methanol, ethanol, propanol and butanol, in particular n-propanol or isopropanol.
- the polypeptide / print molecule mixture is freeze-dried.
- the polypeptide / print molecule mixture is both precipitated and freeze-dried.
- the imprinted conformation of the polypeptide is covalently fixed in a further step by copolymerization via the polymerizable groups containing unsaturated bonds.
- the copolymerization is preferably initiated by UV radiation, peroxides or radical initiators (thermal or by UV radiation).
- Azobis compounds such as ⁇ , ⁇ '-azo-isobutyronitrile (AIBN) or 2,2'-azo-bis- (2,4-dimethyl) valeric acid nitrile (ABDV) are preferably used as radical initiators.
- AIBN or ABDV are particularly preferably used as radical initiators and the copolymerization is initiated by means of UV radiation.
- the process according to the invention is further characterized in that a crosslinker is used for the covalent fixation of the conformation of the derivatized imprint polypeptides obtained after the precipitation and / or freeze-drying.
- Crosslinking compounds containing unsaturated bonds or mixtures thereof selected from the group consisting of analogs or derivatives of the general formula
- and R 3 are, independently of one another, hydrogen atoms, cyclic or linear, unsaturated or saturated, substituted or unsubstituted alkyl, carboxyl, carboxyalkyl or alkyl ether radicals having preferably up to 10, in particular 1 or 2, carbon atoms or substituted or unsubstituted aryl radicals and the radical R 2 a cyclic or linear, saturated or unsaturated, substituted or unsubstituted alkyl or alkyl ether radical with preferably up to 10, in particular 1 or 2 Is carbon atoms or an aryl radical and n has a value of preferably up to 10 and in particular 1 or 2.
- radicals R 1 and R 3 are preferably hydrogen atoms. In a further preferred embodiment, the radicals R, and / or R 3 can contain more than 10 carbon atoms.
- n has a value greater than 10.
- Examples are olefinic polyethylene glycols or olefinic derivatives of polyacrylamides.
- radicals RR 2 and R 3 are unsaturated and / or substituted radicals, they have the meaning given for the general formula (I). In addition to the substituents mentioned, hydroxyl and / or amino radicals can be present.
- crosslinkers used are preferably selected from the group consisting of divinylalkyl and divinylaryl compounds, in particular divinylbenzene or 1,5-hexadiene.
- the crosslinkers or mixtures thereof containing unsaturated bonds are selected from the group consisting of analogs or derivatives of the general formula (III)
- radical R b R 2 and R 3 and n have the meaning given above for the general formula (II) and the radical X is an oxygen atom or the group -NH.
- the crosslinkers are preferably selected from ethylene glycol dimethacrylate, N, N'-methylene-bis-acrylamide, diitaconic acid alkylamides and diitaconic acid arylamides.
- the copolymerization is carried out in cyclohexane, toluene, acetone, alkanoia, ethyl acetate, tetrahydrofuran, chloroform or mixtures thereof, in particular in cyclohexane, ethylene glycol dimethacrylate, divinylbenzene, diitaconic acid alkylamides, arylamides, divinylalkyl or aryl compounds being used as crosslinkers .
- the derivatized imprint polypeptide obtained after the precipitation step is in an organic solvent suspended, one or more of the above-mentioned crosslinkers added, the radical initiator, for example azobis compounds ( ⁇ , ⁇ '-azo-isobutyronitrile (AIBN) or 2,2'-azo-bis- (2,4-dimethyl) - valeric acid nitrile (ABDV)) , added and the copolymerization started by UV radiation.
- the radical initiator for example azobis compounds ( ⁇ , ⁇ '-azo-isobutyronitrile (AIBN) or 2,2'-azo-bis- (2,4-dimethyl) - valeric acid nitrile (ABDV)
- AIBN ⁇ '-azo-isobutyronitrile
- ABDV 2,2'-azo-bis- (2,4-dimethyl) - valeric acid nitrile
- the polypeptide conformation is fixed covalently via the previously introduced olefinic double bonds; the
- the covalently cross-linked imprint polypeptide which is fixed and stabilized in its conformation, can be used under many conditions under which it would have been unstable without the selective introduction of residues and the copolymerization, e.g. in aqueous solvent systems.
- a polypeptide is derivatized by the covalent introduction of polymerizable groups containing unsaturated bonds.
- the polypeptide can be selected from the group mentioned above.
- the derivatization can be carried out as described for step (A) above.
- the polypeptide is then imprinted (embossed) with a print molecule in an aqueous medium.
- the print molecule can be selected as described above.
- a native enzyme is preferably used as the polypeptide and the substrate of this enzyme is used as the print molecule.
- the copolymerization is then carried out in an aqueous solvent using a crosslinker which is copolymerizable with the polymerizable groups containing unsaturated bonds.
- the copolymerization is carried out as described above for step (D).
- the crosslinkers are preferably selected from the groups described above.
- the crosslinker is chosen so that it is soluble in the aqueous solvent.
- N, N'-methylene-bisacrylamide is preferably used as the crosslinker.
- ⁇ -Chymotrypsin is dissolved in 10 ml of 0.05 M potassium phosphate buffer pH 7.8 with stirring. Itaconic anhydride is then added in spatula tips at room temperature. The decrease in the pH of the reaction solution which occurs each time the anhydride is added is corrected by adding 5 M NaOH. After the addition of the anhydride has ended, the mixture is stirred for a further 1 hour and the pH is again corrected. To separate the low molecular weight components, the reaction solution obtained is buffered in 0.05 M potassium phosphate buffer pH 7.8 using PD 10 gel filtration columns (Pharmacia) and finally lyophilized.
- the enzymatic activity of ⁇ -chymotrypsin is determined by means of the hydrolysis of benzoyl-L-tyrosine ethyl ester in a photometric test.
- the extent of the modification of the functional groups is determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS assay) (Habeeb A.F.S.A. (1966), Anal. Biochem. 14, 328-336).
- Tab. 1 shows the reaction batches, the extent of the modification of the functional groups and the enzymatic activities of the ⁇ -chymotrypsin derivatives in relation to the free enzyme.
- the ⁇ -glucosidase is dissolved in 10 ml of 0.05 M citric acid-phosphate buffer pH 6.0 with stirring.
- D (+) - glucose can be added to stabilize the enzyme.
- Itaconic anhydride is then added in spatula tips at room temperature.
- the decrease in the pH of the reaction solution which occurs each time the anhydride is added is corrected by adding 5 M NaOH.
- the mixture is stirred for a further 1 hour and the pH is again corrected.
- the reaction solution obtained is buffered in 0.05 M citric acid-phosphate buffer pH 6.0 using PD 10 gel filtration columns (Pharmacia) and finally lyophilized.
- the enzymatic activity of the ⁇ -glucosidase is determined by means of the hydrolysis of 4-nitrophenyl- ⁇ -D-glucopyranoside in a photometric test.
- the extent of the modification of the functional groups is determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS assay).
- Table 2 shows the reaction batches, the extent of the modification of the functional groups and the enzymatic activities of the ⁇ -glucosidase derivatives in relation to the free enzyme.
- glucose was added as an additive. In the presence of 3M glucose, 99% residual activity could be obtained after derivatization.
- ⁇ -N-acetyl- (L) - amino acids are dissolved in 5 ml of 0.05 M citric acid-phosphate buffer pH 7.5, mixed with itaconic anhydride while stirring and incubated for 1 hour at room temperature.
- the TNBS assay is performed with a sample of each in the buffer dissolved ⁇ -N-acetyl- (X) -amino acid and with the ⁇ -N-acetyl- (Z.) - amino acid incubated with itaconic anhydride.
- Example 4 Use of the cross-linked imprint polypeptide (VIP) in an aqueous medium
- the VIP is suspended in phosphate buffer (10 mM), pH 7.8 and preincubated in a thermal shaker at 27 ° C for approx. 10 min.
- the reaction is started by adding the substrate, the N-acetyl- () -tryptophanethylester (10 mM).
- the VIP showed a specific (D) - ester hydrolysis activity of 0.04 ⁇ mol / (min • g).
- the native non-printed, the non-cross-linked imprinted and the non-imprinted cross-linked ⁇ -chymotrypsin had no (-D) ester hydrolysis activity.
- the VIP was incubated for 1 h in a phosphate buffer (10 mM), pH 7.8 at 25 ° C. After the batch had been lyophilized, the VIP was suspended in cyclohexane. After adding N-Ac- (D) -Trp (10 mM) and 20% (v / v) ethanol, the N-acetyl- () - tryptophanethyl ester synthesis was started at 25 ° C. in a thermoshaker. The VIP showed one specific (Z ) ) ester hydrolysis activity of 0.02 ⁇ mol / (min • g). The imprinted property was therefore not lost in the aqueous solvent system.
- Example 5B As in Example 5A. described, but using N-Ac- (D) -tyrosine instead of N-Ac - (. D) -Trp.
- bovine serum albumin 50 mg bovine serum albumin are dissolved in 10 ml 0.05 M potassium phosphate buffer pH 5.5. At room temperature, spatula tips of itaconic anhydride are then added with stirring. The decrease in the pH of the reaction solution which occurs each time the anhydride is added is corrected by adding 5 M NaOH. After the addition of the anhydride has ended, the mixture is stirred for a further 1 hour and the pH is again corrected. To separate the low molecular weight components, the reaction solution obtained is buffered in 0.05 M potassium phosphate buffer pH 5.5 using PD 10 gel filtration columns (Pharmacia) and finally lyophilized. The extent of the modification of the functional groups is determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS assay).
- TNBS assay 2,4,6-trinitrobenzenesulfonic acid
- the aqueous solution contains L-malic acid in a concentration of 0.5 M.
- the solution is cooled at pH 5.5 to 0 ° C and 4 ml of 1-propanol, which has been cooled to -20 ° C, are added.
- the resulting precipitate is centrifuged, washed with 10 ml of 1-propanol and finally lyophilized.
- the copolymerization is started by adding 0.5 ml of an ammonium peroxodisulfate solution (5% w / v) and 0.5 ml of a 3-dimethylaminopropionitrile solution (5% v / v). After a reaction time of 5 hours, the copolymer is first in a pleated filter with 0.5 liters of 0.5 M NaCl solution and then with 2 liters of distilled H 2 O. washed until salt-free. The copolymer is taken up in 0.05 M citric acid-phosphate buffer pH 6.0 and then lyophilized.
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Abstract
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP97931724A EP0912605A1 (en) | 1996-07-05 | 1997-07-07 | Imprint polypeptides covalently cross-linked and having a fixed and stabilised arrangement, process for the preparation and use thereof |
JP10504659A JP2000500777A (en) | 1996-07-05 | 1997-07-07 | Imprinted polypeptides having covalently crosslinked and immobilized and stabilized configurations, methods for their production and their use |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE1996127162 DE19627162C1 (en) | 1996-07-05 | 1996-07-05 | New covalently crosslinked imprint poly:peptide, e.g. enzyme |
DE19627162.2 | 1996-07-05 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1998001466A1 true WO1998001466A1 (en) | 1998-01-15 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DE1997/001448 WO1998001466A1 (en) | 1996-07-05 | 1997-07-07 | Imprint polypeptides covalently cross-linked and having a fixed and stabilised arrangement, process for the preparation and use thereof |
Country Status (5)
Country | Link |
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EP (1) | EP0912605A1 (en) |
JP (1) | JP2000500777A (en) |
CA (1) | CA2259416A1 (en) |
DE (1) | DE19627162C1 (en) |
WO (1) | WO1998001466A1 (en) |
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US6884842B2 (en) | 1997-10-14 | 2005-04-26 | Alnis Biosciences, Inc. | Molecular compounds having complementary surfaces to targets |
DE19839538A1 (en) * | 1998-08-31 | 2000-03-02 | Gluesenkamp Karl Heinz | Manufacture of customized chromatography materials over intelligent surfaces |
JP6338928B2 (en) * | 2014-05-21 | 2018-06-06 | 三栄源エフ・エフ・アイ株式会社 | Quantitative analysis of protein in cochineal dye |
CN109897144B (en) * | 2019-02-28 | 2020-05-19 | 南开大学 | Polypeptide-crosslinked protein molecularly imprinted polymer and preparation method and application thereof |
GB202209432D0 (en) * | 2022-06-28 | 2022-08-10 | Croda Int Plc | Composition, home care formulations, method and use |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2088880A (en) * | 1980-12-04 | 1982-06-16 | Owens Illinois Inc | Process for preparing semisynthetic proteins |
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SE9203913D0 (en) * | 1992-12-28 | 1992-12-28 | Ian A Nicholls | PREPARATION OF POLYMERS THROUGH MOLECULE PRINT USING STEREO AND ENANTIOSELECTIVE SYNTHESIS PRIMED BY NON-COVALENT INTERACTIONS |
-
1996
- 1996-07-05 DE DE1996127162 patent/DE19627162C1/en not_active Expired - Fee Related
-
1997
- 1997-07-07 JP JP10504659A patent/JP2000500777A/en active Pending
- 1997-07-07 EP EP97931724A patent/EP0912605A1/en not_active Withdrawn
- 1997-07-07 CA CA 2259416 patent/CA2259416A1/en not_active Abandoned
- 1997-07-07 WO PCT/DE1997/001448 patent/WO1998001466A1/en not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2088880A (en) * | 1980-12-04 | 1982-06-16 | Owens Illinois Inc | Process for preparing semisynthetic proteins |
Non-Patent Citations (4)
Title |
---|
CHEMICAL ABSTRACTS, vol. 116, no. 15, 13 April 1992, Columbus, Ohio, US; abstract no. 146685, XP002046014 * |
G WULFF: "Molecular imprinting in cross-linked materials with the aid of molecular templates- a way toward artificial antibodies", ANGEWANDTE CHEMIE INTERNATIONAL EDITION., vol. 34, 1995, WEINHEIM DE, pages 1812 - 1832, XP000524863 * |
M STAHL ET AL.: "Induced stereoselectivity and substrate selectivity of bio-imprinted alpha-chymotrypsin in anhydrous organic media", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 113, no. 24, 20 November 1991 (1991-11-20), DC US, pages 9366 - 9368, XP002046013 * |
T KRIEGEL ET AL.: "Yeast phosphofructokinase: kinetic characterization of a substrate-imprinted enzyme conformation", BIOMED. BIOCHIM. ACTA, vol. 50, no. 12, 1991, pages 1159 - 1166 * |
Also Published As
Publication number | Publication date |
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CA2259416A1 (en) | 1998-01-15 |
DE19627162C1 (en) | 1997-08-07 |
JP2000500777A (en) | 2000-01-25 |
EP0912605A1 (en) | 1999-05-06 |
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